Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during pegylated uricase therapy

ABSTRACT

Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response during intravenous PEGylated uricase therapy in gout patients is provided. Routine SUA monitoring can be used to identify patients receiving PEGylated uricase who may no longer benefit from treatment and who are at greater risk for infusion reactions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/906,839, filed on Feb. 27, 2018, now U.S. Pat. No. 10,139,399, whichis a continuation of U.S. application Ser. No. 15/165,318, filed on May26, 2016, which is a continuation of U.S. application Ser. No.13/379,704, filed on Aug. 8, 2012, now U.S. Pat. No. 9,377,454, which isa national stage filing of International Application No.PCT/US2010/040082, filed on Jun. 25, 2010, which claims priority to andbenefit of U.S. Provisional Application No. 61/269,669, filed on Jun.25, 2009, U.S. Provisional Application No. 61/248,698, filed on Oct. 5,2009, and U.S. Provisional Application No. 61/298,718, filed on Jan. 27,2010, the disclosures of which are hereby incorporated by reference asif written herein in their entireties.

FIELD OF THE INVENTION

This invention relates to methods for monitoring immunogenicity andinfusion reactions during PEGylated uricase therapy.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced withinthe text. The disclosure of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled in thereinas of the date of the invention described and claimed herein.

Gout is a chronic disorder of urate metabolism resulting in depositionof monosodium urate crystals in the joints and soft tissues, withaccompanying inflammation and eventually, in some patients, destructive,chronic arthropathy. Gout is the most prevalent form of arthritis in menand is increasing in incidence and prevalence among older persons ofboth genders. Chronic gout refractory to Conventional Therapy (GRT) isan uncommon but severe outcome of progressive gout resulting fromdemonstrated intolerance of or refractoriness to available therapy toprevent urate crystal deposition by reducing and maintaining serum uratelevels in a subsaturating range.

Elevated serum urate is a hallmark biochemical marker of gout.Persistently elevated plasma uric acid (PUA) or serum uric acid (SUA)levels result in deposition of uric acid in joints and soft tissues. Asthe total body burden of uric acid increases, signs and symptoms of goutresult, including arthritis, characterized by recurrent painful goutflares, development of tophi and joint deformities with resultantchronic pain/inflammation and consequent loss of physical function.

The efficacy end point of successful PEGylated uricase therapy isnormalization of serum uric acid levels in CGR patients whilemaintaining low immunogenicity profile and low risk of infusionreactions associated with intravenous injections of PEGylated uricase.However, given that the loss of PEGylated uricase effect and infusionreactions can accompany PEGylated uricase administration, cliniciansshould be advised as to the proper time point at which to discontinuetherapy. Thus, there is a need in the art for new methods to guideclinicians when to discontinue the PEGylated uricase therapy in order tominimize infusion reactions and their associated safety risks.

SUMMARY OF THE INVENTION

The present invention provides for methods of preventing infusionreactions during PEGylated uricase therapy in a patient comprising thesteps of a) administering to said patient PEGylated uricase; b)obtaining a biological sample from said patient;

c) determining uric acid levels in said biological sample; and d)indicating that therapy may be discontinued to prevent infusionreactions when said uric acid level is more than about 4 mg/dl. In oneaspect of the invention, PEGylated uricase therapy may be discontinuedwhen said uric acid level is more than about 5 mg/dl. In another aspectof the invention, PEGylated uricase therapy may be discontinued whensaid uric acid level is more than about 6 mg/dl and in yet anotheraspect of the invention, the PEGylated uricase therapy may bediscontinued when said uric acid level is more than about 7 mg/dl.

In another aspect of the invention, the PEGylated uricase isadministered at a dosage of about 8 mg every 2 weeks. In one embodiment,the PEGylated uricase is administered at a dosage of about 8 mg every 3weeks. In another embodiment, the PEGylated uricase is administered at adosage of about 8 mg every 4 weeks. In yet another embodiment, thePEGylated uricase is administered at a dosage of about 4 mg every 2weeks. In yet another embodiment, the PEGylated uricase is administeredat a dosage of about 12 mg every 4 weeks.

The methods of the present invention provides for biological sampleselected from the group consisting of blood, serum and plasma. In oneembodiment, said uric acid levels in said biological sample aredetermined at least 2 hours after administration as defined in step (a).In another embodiment, said uric acid levels in said biological sampleare determined at least 6 hours after administration as defined in step(a). In yet another embodiment, said uric acid levels in said biologicalsample are determined at least 24 hours after administration as definedin step (a). In yet another embodiment, said uric acid levels in saidbiological sample are determined 2 weeks after administration as definedin step (a). And in yet another embodiment, said uric acid levels insaid biological sample are determined 4 weeks after administration asdefined in step (a).

The methods of the present invention relate to patients suffering fromgout. In one embodiment, said gout is refractory. In another embodiment,said gout is chronic or tophaceous. In yet another embodiment, thePEGylated uricase is administered intravenously.

The methods of the present invention predict whether a patient treatedwith PEGylated uricase will develop infusion reaction, wherein themethod comprises the steps of: a) administering to said patientPEGylated uricase; b) obtaining a biological sample from said patient;c) determining uric acid levels in said biological sample; and d)indicating that uric acid level is associated with a lower likelihood ofinfusion reaction when said level is maintained at less than about 4mg/dl or indicating that said determined uric acid level is associatedwith a higher likelihood of infusion reaction at a time point when saidlevel is measured at least about 4 mg/dl.

In one aspect of the invention, said uric acid level is associated witha lower likelihood of infusion reaction when said level is maintained atless than about 5 mg/dl or said determined uric acid level is associatedwith a higher likelihood of infusion reaction at a time point when saidlevel is measured at least about 5 mg/dl. In another aspect of theinvention, said uric acid level is associated with a lower likelihood ofinfusion reaction when said level is maintained at less than about 6mg/dl or said determined uric acid level is associated with a higherlikelihood of infusion reaction when said level is measured at leastabout 6 mg/dl. In yet another aspect of the invention, said uric acidlevel is associated with a lower likelihood of infusion reaction whensaid level is maintained at less than about 7 mg/dl or said uric acidlevel is associated with a higher likelihood of infusion reaction at atime point when said uric acid level is measured at least about 7 mg/dl.

In another aspect of the invention, the uric acid levels in saidbiological sample are determined at least 3 days after theadministration as defined in step (a). In another aspect of theinvention, the uric acid levels in said biological sample are determinedat least 1 week after the administration as defined in step (a). Inanother aspect of the invention, the uric acid levels in said biologicalsample are determined at least 2 weeks after the administration asdefined in step (a). In another aspect of the invention, the uric acidlevels in said biological sample are determined at least 4 weeks afterthe administration as defined in step (a).

The methods of the present invention predict whether a patient treatedwith PEGylated uricase will develop antibody-mediated PEGylated uricaseclearance without measuring anti-PEGylated uricase and anti-PEGantibodies titer, wherein the method comprises the steps of a)administering to said patient PEGylated uricase; b) obtaining abiological sample from said patient; c) determining uric acid levels insaid biological sample; and d) indicating that uric acid level isassociated with a lower likelihood of antibody-mediated PEGylateduricase clearance when said level is maintained at less than about 4mg/dl or indicating that said determined uric acid level is associatedwith a higher likelihood of antibody-mediated PEGylated uricaseclearance at a time point when said uric acid level is measured at leastabout 4 mg/dl.

In one aspect of the invention, said uric acid level is associated witha lower likelihood of antibody-mediated PEGylated uricase clearance whensaid level is maintained at less than about 5 mg/dl or indicating thatsaid determined uric acid level is associated with a higher likelihoodof antibody-mediated PEGylated uricase clearance at a time point whensaid uric acid level is measured at least about 5 mg/dl. In anotheraspect of the invention, said uric acid level is associated with a lowerlikelihood of antibody-mediated PEGylated uricase clearance when saidlevel is maintained at less than about 6 mg/dl or indicating that saiddetermined uric acid level is associated with a higher likelihood ofantibody-mediated PEGylated uricase clearance at a time point when saiduric acid level is measured at least about 6 mg/dl. In yet anotheraspect of the invention, said uric acid level is associated with a lowerlikelihood of antibody-mediated PEGylated uricase clearance when saidlevel is maintained at less than about 7 mg/dl or indicating that saiddetermined uric acid level is associated with a higher likelihood ofantibody-mediated PEGylated uricase clearance at a time point when saiduric acid level is measured at least about 7 mg/dl.

In another aspect of the invention, the uric acid levels in saidbiological sample are determined at least 3 days after theadministration as defined in step (a). In another aspect of theinvention, the uric acid levels in said biological sample are determinedat least 1 week after the administration as defined in step (a). Inanother aspect of the invention, the uric acid levels in said biologicalsample are determined at least 2 weeks after the administration asdefined in step (a). In another aspect of the invention, the uric acidlevels in said biological sample are determined at least 4 weeks afterthe administration as defined in step (a).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mean anti-pegloticase antibody titer in patients receivingpegloticase every 2 Weeks.

FIG. 2 shows time-concentration profile for pegloticase every 2 weekadministration.

FIG. 3 shows relationship between antibody titer and AUC pegloticaseevery 2 week administration.

FIG. 4 shows SUA value at first detected loss response pegloticase every2 weeks.

FIG. 5 shows anti-pegylated uricase antibody titer at time of loss ofresponse pegloticase every 2 weeks.

FIG. 6 shows time-concentration profile for pegloticase every 4 weekadministration.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In accordance with this detailed description, the followingabbreviations and definitions apply. It must be noted that as usedherein, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

It had been surprisingly discovered that monitoring SUA levels predictsantibody-mediated loss of response and the majority of infusionreactions during PEGylated uricase therapy. It has been found that mostinfusion reactions occurred after loss of SUA response. Therefore,routine monitoring of SUA can be used to prospectively identify patientsreceiving PEGylated uricase who no longer benefit from treatment and areat a greater risk for infusion reactions.

The term “therapeutic efficacy” as used herein refers to theeffectiveness of a particular treatment regimen. Specifically,therapeutic efficacy is defined by achieving serum urate levels less orabout 6 mg/dl. This includes a balance of efficacy, toxicity (e.g., sideeffects and patient tolerance of a formulation or dosage unit), patientcompliance, and the like.

The terms “treating,” “treatment,” and the like are used herein to referto obtaining a desired pharmacological and physiological effect. Theeffect can be prophylactic in terms of preventing or partiallypreventing a disease, symptom, or condition thereof and/or can betherapeutic in terms of a partial or complete cure of a disease,condition, symptom, or adverse effect attributed to the disease. Theterm “treatment,” as used herein, covers any treatment of a disease in amammal, such as a human, and includes: (a) preventing the disease fromoccurring in a patient which can be predisposed to the disease but hasnot yet been diagnosed as having it, i.e., causing the clinical symptomsof the disease not to develop in a patient that can be predisposed tothe disease but does not yet experience or display symptoms of thedisease; (b) inhibiting the disease, i.e., arresting or reducing thedevelopment of the disease or its clinical symptoms; and (c) relievingthe disease, i.e., causing regression of the disease and/or its symptomsor conditions. Treating a patient's suffering from disease related topathological inflammation is contemplated. Preventing, inhibiting, orrelieving adverse effects attributed to pathological inflammation overlong periods of time and/or are such caused by the physiologicalresponses to inappropriate inflammation present in a biological systemover long periods of time are also contemplated.

As used herein the term “immunogenicity” refers to the induction of animmune response by an injected preparation of PEG-modified or unmodifieduricase (the antigen), while “antigenicity” refers to the reaction of anantigen with preexisting antibodies. Collectively, antigenicity andimmumunogenicity are referred to as “immunoreactivity.” In previousstudies of PEGylated uricase, immunoreactivity is assessed by a varietyof methods, including: 1) the reaction in vitro of PEGylated uricasewith preformed antibodies; 2) measurements of induced antibodysynthesis; and 3) accelerated clearance rates of PEGylated uricase afterrepeated injections.

As used herein the term “infusion reaction” is an undesired andunintended effect of a PEGylated uricase occurring within 2 hours afterthe PEGylated uricase or placebo infusion that cannot be reasonablyattributed to another cause. In particular, an adverse drug reactionoccurs at doses used for prophylaxis, diagnosis or therapy.

The PEGylated uricase conjugates of the present invention are useful forlowering the levels of uric acid in the body fluids and tissues ofmammals, preferably humans, and can thus be used for treatment ofelevated uric acid levels associated with conditions including gout,tophi, renal insufficiency, organ transplantation and malignant disease.PEGylated uricase conjugates can be injected into a mammal havingexcessive uric acid levels by any of a number of routes, includingintravenous, subcutaneous, intradermal, intramuscular andintraperitoneal routes.

In one embodiment, PEGylated uricase is administered in apharmaceutically acceptable excipient or diluent at 8 mg every twoweeks. In another embodiment, PEGylated uricase can be administered at 8mg every four weeks. In yet another embodiment, PEGylated uricase can beadministered at 8 mg every three weeks.

In the other aspect of the invention, PEGylated uricase can beadministered at 4 mg every two weeks. In yet another aspect of theinvention, PEGylated uricase can be administered at 12 mg every fourweeks.

Pharmaceutical formulations containing PEGylated uricase can be preparedby conventional techniques, e.g., as described in Gennaro, A R (Ed.)(1990) Remington's Pharmaceutical Sciences, 18th Edition Easton, Pa.:Mack Publishing Co. Suitable excipients for the preparation ofinjectable solutions include, for example, phosphate buffered saline,lactated Ringer's solution, water, polyols and glycerol. Pharmaceuticalcompositions for parenteral injection comprise pharmaceuticallyacceptable sterile aqueous or non-aqueous liquids, dispersions,suspensions, or emulsions as well as sterile powders for reconstitutioninto sterile injectable solutions or dispersions just prior to use.These formulations can contain additional components, such as, forexample, preservatives, solubilizers, stabilizers, wetting agents,emulsifiers, buffers, antioxidants and diluents.

PEGylated uricase can also be provided as controlled-releasecompositions for implantation into an individual to continually controlelevated uric acid levels in body fluids. For example, polylactic acid,polyglycolic acid, regenerated collagen, poly-L-lysine, sodium alginate,gellan gum, chitosan, agarose, multilamellar liposomes and many otherconventional depot formulations comprise bioerodible or biodegradablematerials that can be formulated with biologically active compositions.These materials, when implanted or injected, gradually break down andrelease the active material to the surrounding tissue. For example, onemethod of encapsulating PEGylated uricase comprises the method disclosedin U.S. Pat. No. 5,653,974, which is hereby incorporated by reference.The use of bioerodible, biodegradable and other depot formulations isexpressly contemplated in the present invention. The use of infusionpumps and matrix entrapment systems for delivery of PEGylated uricase isalso within the scope of the present invention. PEGylated uricase canalso advantageously be enclosed in micelles or liposomes. Liposomeencapsulation technology is well known in the art. See, e.g., Lasic, D,et al., (Eds.) (1995) Stealth Liposomes. Boca Raton, Fla.: CRC Press.

The uricase used in PEGylated uricase can comprise a mammalian uricaseamino acid sequence truncated at the amino terminus or the carboxyterminus or both the amino and carboxy termini by about 1-13 amino acidsand can further comprise an amino acid substitution at about position46. The truncated uricase can further comprise an amino terminal aminoacid, wherein the amino terminal amino acid is alanine, glycine,proline, serine, or threonine as described in co-pendingPCT/US2006/013660 and U.S. provisional application Ser. No. 60/670,573,which are hereby incorporated herein by reference in their entireties.

Phase 3 study was completed as indicated in the Examples. In one aspectof the invention, normalization of uric acid of at least about 3.5 mg/dLwas selected as the primary outcome measure to reflect thepharmacodynamic effect of PEGylated uricase. In another aspect of theinvention, normalization of uric acid of at least about 4.0 mg/dL wasselected as the primary outcome measure to reflect the pharmacodynamiceffect of PEGylated uricase. In yet another aspect of the invention,normalization of uric acid of at least about 5.0 mg/dL was selected asthe primary outcome measure to reflect the pharmacodynamic effect ofPEGylated uricase. In yet another aspect of the invention, normalizationof uric acid of at least about 6.0 mg/dL was selected as the primaryoutcome measure to reflect the pharmacodynamic effect of PEGylateduricase. In another aspect of the invention, normalization of uric acidof at least about 7.0 mg/dL was selected as the primary outcome measureto reflect the pharmacodynamic effect of PEGylated uricase.

It is know that persistently elevated plasma uric acid (PUA) or serumuric acid (SUA) levels result in deposition of uric acid in joints andsoft tissues. As the total body burden of uric acid increases, signs andsymptoms of gout result, including arthritis, characterized by recurrentpainful gout flares, development of tophi and joint deformities withresultant chronic pain/inflammation and consequent loss of physicalfunction.

PEGylated uricase 8 mg q2 wk results in marked decreases in uric acid(PUA and SUA) which is associated with complete resolution of tophi insome patients and decreased tender joint counts. Treatment is alsoassociated with a decrease in the incidence and frequency of gout flaresafter 3 months of therapy compared with placebo, with continuedreductions in flare incidence and frequency with long termadministration, up to at least 18 months. These benefits occur inpatients with chronic and often severe disease who have no othercurrently available therapy. Persistent responders are those patientswho maintain lowered SUA values in response to repeated PEGylateduricase infusions. Maintenance of lowered SUA values is associated withno or low anti-PEGylated uricase antibody response (titers <2430).

The relationship between measured plasma uric acid (PUA) and serum uricacid (SUA) values was evaluated from serial samples from all patients inphase 3 studies. The rationale for this evaluation related to the use ofPUA as the measure for the primary endpoint for all PEGylated uricasetrials while SUA is used in clinical practice. The handling andprocessing of samples for PUA determination is much more involved, andthis processing was performed at low temperature and utilizedtrichloroacetic acid to inactivate and precipitate PEGylated uricase sothe drug did not continue to oxidize uric acid. Nevertheless, theexperimental results unequivocally show a close correlation between bothuric acid values at all time points and irrespective of the uric acidvalues.

An infusion reaction was defined as any adverse event that occurredduring or within 2 hours after the PEGylated uricase or placebo infusionthat could not be reasonably attributed to another cause. Although therewas protocol-specified infusion reaction prophylatic treatment, infusionreactions occurred in 26% of patient treated with PEGylated uricase q2wk and 40% with PEGylated uricase q4 wk.

Anti-PEGylated uricase antibodies were observed in about 90% of patientstreated with PEGylated uricase. Antibodies at higher titers (>1:2430)were associated with increased clearance of PEGylated uricase and lossof PEGylated uricase activity, but high titers were frequently notdetected until some time after uric acid levels were increased,sometimes lagging by several weeks after the loss of PEGylated uricaseresponse. Patients who initially responded to PEGylated uricase and lostresponse at later time points were referred to as transient responders,in contrast to patients who maintained urate lowering activity ofPEGylated uricase throughout the study and were termed persistentresponders. The rise in PUA precedes the evidence of higher titers ofantibodies.

Patients who developed high antibody titers (but not lower titers) had ahigh likelihood of loss of PUA response. The evidence of a transientresponse was clear in all patients by month 4 following initiation oftherapy. The clinical effects of immunogenicity are easily detected byregular monitoring of SUA levels during the first few months of therapy.Although those patients who developed higher titer antibodies had ahigher incidence of infusion reactions, there was no clear relationshipbetween antibody titer and severity of infusion reactions.

The results herein indicate the development of high titer anti-PEGylateduricase antibodies and anty titer of anti-PEG explains the loss of theSUA/PUA response. In patients that eventually develop higher titers ofantibodies to PEGylated uricase there a higher risk of infusionreactions. Importantly, most infusion reactions occur after the loss ofSUA/PUA response and, as a result, careful monitoring of SUA can avoidunnecessary dosing and also prevent the majority of infusion reactions.The loss of effect in most transient responders occurs within the first4 months, so monitoring serum uric acid during that time period iscritical. Finally, the loss of effect of PEGylated uricase canfrequently occur before the rise in anti-PEGylated uricase antibodytiter, so that there is no correlation between the titer ofanti-PEGylated uricase antibody, or the presence of any titer anti-PEGantibody, before or at the time of loss of a SUA/PUA response. The lackof association between antibody titer and the SUA/PUA response confirmsthe ineffectiveness of monitoring antibody titers during PEGylateduricase therapy of patients with treatment failure gout.

Example 1—Immunogenicity and Infusion Reaction Profiles of PegloticaseIntravenous Administration at 8 mg Every 2 Weeks

Material, Methods and Design of Clinical Study.

Investigational Drug

Pegloticase, a PEGylated uricase used in this example, consists of arecombinant mammalian uricase (primarily porcine, with C-terminalsequence from baboon uricase), conjugated with multiple strands ofmonomethoxy PEG of average molecular weight 10 kDa (10 K mPEG) persubunit of tetrameric enzyme (Kelly S J, et al. J Am Soc Nephrol 2001,12:1001-1009; and Ganson N J, et al. Arthritis Res Ther 2005, 8(1):R12).

Phase III Study Design.

Patients:

Multi-center (45 sites), replicate, double-blind, placebo-controlled,studies were performed in patients with symptomatic gout.

All patients received an intravenous (i.v.) infusion (pegloticase orplacebo) every 2 weeks. Treatment groups consisted of placebo (N=43),pegloticase 8 mg i.v. every 2 weeks (q2 wks) (N=84).

All patients reported a medical history in which allopurinol therapy wascontraindicated (e.g., history of hypersensitivity, intolerance, ortoxicity) or had not been effective, defined as failure to normalize SUAwith ≥3 months allopurinol treatment at the maximum labeled dose (800mg/day) or at a medically appropriate lower dose based on toxicity ordose-limiting co-morbidity. The major exclusion criteria at entryincluded: unstable angina, uncontrolled arrhythmia, non-compensatedcongestive heart failure, uncontrolled hypertension (above 150/95 mmHg),dialysis, organ transplant recipient, pregnancy and other.

For these experiments, all patients discontinued all urate-loweringtherapies ≥one week prior to randomization, and refrained from usingsuch agents throughout the study.

All patients received prophylaxis for infusion reactions (IR): oralfexofenadine (60 mg evening prior and immediately before infusion), andacetaminophen (1000 mg) and hydrocortisone IV (200 mg) prior to eachinfusion. Study medication was administered in 250 mL saline over 2 to 4hours total infusion time.

Immunogenicity

The qualitative and quantitative ELISA assays used for study sampleanalysis were validated to Good Laboratory Practices following acceptedimmunology assay guidance (Mire-Sluis et al). Samples for antibodydetermination using ELISA assays were collected from all patients atbaseline and at Weeks 3, 5, 9, 13, 17, 21 and 25 after initiation oftreatment with pegloticase or placebo.

Detection of Anti-Pegloticase Antibody.

For determination of total pegloticase antibodies, study samples werediluted 1/30 in assay buffer and assayed using microtiter ELISA platewells coated with either pegloticase or PEG. A human serum containingpegloticase antibodies was used as a positive control for detection oftotal pegloticase antibody as well as IgM and IgG antibodies. Thecombination of rabbit anti-human IgM and IgG was used as secondaryantibodies, whereas each individually was employed for assay of IgM andIgG anti-pegloticase antibodies, respectively (Sigma, St. Louis, Mo.)

For these experiments, horseradish peroxidase-conjugated mousemonoclonal antibody to rabbit IgG was used for detection. Microtiterplate wells coated with purified human IgG and IgM served asimmunoglobulin positive controls for the binding of anti-human IgG andanti-human IgM secondary antibodies.

Drug interference was determined to be 300 μg/mL which is much higherthan the measured circulating pegloticase concentration determined inthe study samples. Therefore, circulating pegloticase would not beanticipated to interfere with the measurement of anti pegloticaseantibodies.

Properties of the Anti-Pegloticase Antibodies.

For the majority of samples from the phase 3 patients, the antibodyresponse involved both IgM and IgG antibodies.

Detection of Anti-Pegloticase Antibodies.

For these experiments, the anti-pegloticase analysis methodologyparallels the general method for the anti-pegloticase antibody assay,with the exception that a surrogate positive control was used for theinitial study sample analyses. This positive control consisted of amixture of mouse monoclonal anti-PEG IgG 1 and anti-PEG IgM antibodies,added to pooled human serum and diluted 1/10 in blocker casein in PBS. Ahuman positive control was introduced in the assay towards the end ofthe study sample analysis. For these experiments, the assay sensitivitywas 500 ng/mL and is also reflected in a low false detection rate of8.6%.

Safety Evaluations—Infusion Reactions.

For these experiments, infusion reactions were defined as any adverseevent that occurred during or within 2 hours after the infusion ofblinded study medication that could not be reasonably attributed toother causes. Infusion reactions occurred during the infusion ofpegloticase and placebo. Signs and symptoms of serious infusionreactions included: dyspnea, hypotension, hypertension, swelling,brochospasm, chest pain, nausea, vomiting and abdominal pain andcramping.

As shown in FIG. 1, at all time points after dosing, the persistentresponders in the q2 wk group had lower mean anti-pegloticase antibodytiters compared to the transient responders. For example, it wasobserved that patients with anti-pegloticase antibody titer <1:810 atany time during the study were associated with persistent response.Thus, 68% of the q2 week persistent responders had titers that neverexceeded a titer of 1:810. On the other hand, only 23% of the q2 weektransient responders had titers <1:810. Therefore, low titer wasassociated with persistent response.

Anti-Pegloticase Antibody Effects on Pegloticase Pharmacokinetics andPharmacodynamics.

As shown in FIG. 2, the pharmacokinetics of pegloticase administeredevery 2 weeks is significantly influenced by the presence of pegloticaseantibodies. Persistent responders had higher pegloticase peakconcentrations (Cmax) in both groups compared to transient responders.As shown in FIG. 2, transient responders in the q2 wk dose group showeddecreased peak pegloticase concentrations after week 3. Further, by week15, the transient responders had pegloticase concentrations that werebelow the level of detection (0.6 μg/mL). Persistent responders in theq2 wk group had pegloticase concentrations in the range of 0.5-0.7μg/mL.

As shown in FIG. 3, in transient responders, the increasedanti-pegloticase antibody titers were associated with markedly decreasedpegloticase levels as assessed by the area under the time-concentrationcurve (AUC) compared with the pegloticase levels in the persistentresponders. While there is an association between loss of response anddevelopment of higher pegloticase titers, loss of response could occurcontemporaneously or even before the rise in antibody titer. Therefore,titer determinations are not predictive of loss of pegloticase response.

Anti-Pegloticase Antibody Effects on SUA/PUA Response: SUA/PUA as aSurrogate for Physiologically Relevant Anti-Pegloticase Antibodies.

It was further investigated when the SUA increase above 6 mg/dL occurredin the transient responder group following administration of pegloticase8 mg q2 wks. Each point in the top panel of FIG. 4 represents the firstmeasured SUA value that exceeded the threshold value of 6 mg/dL and thetime at which this event occurred for each individual transientresponder in the q2 wks group. FIG. 5 shows the corresponding antibodytiters that were measured at or before the time of loss of uricaseresponse. However, given that treatment with pegloticase q2 wks resultsin SUA values that are generally less than 3 mg/dL, the threshold SUAvalue representing a loss of pegloticase response can be set at an evenlower value, for example between about 3.5 mg/dL to about 7 mg/dl. Butas the rise in SUA due to loss of pegloticase response is generallyrapid, 6 mg/dL is one of the accepted thresholds for control of uricacid by urate lowering drugs. However, 4 mg/di, and 5 mg/di, can be usedsuccessfully in these experiments as a threshold value for control ofuric acid by urate lowering drugs.

At the time of loss of SUA normalization in the q2 wk group, i.e., whenSUA exceeded 6 mg/dL, there was a wide range of ant-pegloticase antibodytiters so that there appeared no threshold antibody titer thatcorresponded to this loss of response, as shown in FIG. 5. Specifically,at the time of loss of urate response, mean anti-pegloticase antibodytiters were 1:3032 for the q2 wk group as compared to a mean highesttiter of 1:686 for the q2 wk persistent responders.

Infusion Reaction and Loss of SUA Normalization.

Most patients (90.9%) had infusion reactions after pegloticase activitywas lost, that is when SUA values were greater than or equal to 5 mg/dL(Table 1).

TABLE 1 SUA Category At Time of Infusion Reaction in Patients Receivingpegloticase Every 2 Weeks pegloticase 8 mg q2 wk Placebo SUA Categoryn/N (%) n/N (%) Number of Patients 20/22 (90.9) 2/43 (4.7) with IR whenSUA ≥ 5 mg/dL Number of Patients 1/22 (4.5) 0 with IR when SUA < 5 mg/dLNumber of Patients 1/22 (4.5) 0 with IR at First Dose

As shown in Table 1, in q2 wk group, 90.9% of infusion reactions wouldhave been prevented if pegloticase therapy was discontinued at the timepoint when SUA≥5 mg/dL.

In summary, anti-pegloticase antibodies have direct effects on thepharmacokinetic and pharmacodynamic properties of pegloticase andexplain the transient effect of pegloticase in the patients who developphysiologically-relevant antibodies. Although the increased clearance ofpegloticase with the resultant loss of SUA/PUA response is mediated byanti-pegloticase antibodies, the initiation of increased clearance doesnot correlate with the anti-pegloticase antibody titer. Therefore,measurement of anti-pegloticase antibody titers is not predictive of theloss of the SUA/PUA response, whereas monitoring SUA/PUA is a very goodsurrogate for measuring the development of anti-pegloticase antibodiesthat cause increased clearance of administered pegloticase. Mostimportantly, monitoring SUA values, particularly during the first 4months after initiating treatment with pegloticase, and stoppingtreatment with pegloticase when SUA values rise to levels greater thanabout 3.5 to 4 mg/dL is a simple method for identifying individuals wholose response to pegloticase and are at higher risk of experiencing aninfusion reaction.

Example 2—Immunogenicity and Infusion Reaction Profiles of Phase IIIClinical Study: Pegloticase Intravenous Administration at 8 mg Every 4Weeks

Clinical Study Using Infusion of Pegloticase.

A multicenter, randomized, double-blind placebo controlled clinicalstudy was carried out as indicated in Example 1 above. Patients withhyperuricemia and gout received pegloticase 8 mg intravenously every 4weeks (N=84) or placebo (N=43). Treatment was administered for 24 weeks.

Patients must have discontinued any uric acid-lowering agents for atleast one week prior to receiving study drug, and refrain from usingsuch agents throughout the study.

Anti-pegloticase antibodies were detected in 88% of patients in thepegloticase 8 mg q4 wk and in only 15% of the placebo group.

As shown in FIG. 6, the pharmacokinetics of pegloticase administeredevery 4 weeks is significantly influenced by the presence ofanti-pegloticase antibodies. Persistent responders had higherpegloticase peak concentrations (Cmax) in both groups compared totransient responders.

Table 2 shows that most patients (76.5%) who had an infusion reactionhad SUA values at or above 6 mg/dL at the time the infusion reactionoccurred. These infusion reactions could have been prevented ifpegloticase was discontinued at the time point that SUA values were ≥6mg/dL. Four patients had infusion reactions when SUA was less than 6mg/dL and four patients who had an infusion reaction at first dose; noneof these infusion reactions could have been prevented by monitoring SUAvalues.

TABLE 2 SUA Category at Time of Infusion Reaction in Patients Receivingpegloticase Every 4 Weeks pegloticase 8 mg q4 wk Placebo SUA Categoryn/N (%) n/N (%) Number of Patients 26/34 (76.5)  2/43 (4.7) with IR whenSUA ≥ 6 mg/dL Number of Patients 4/34 (11.8) 0 with IR when SUA < 6mg/dL Number of Patients 4/34 (11.8) 0 with IR at First Dose

What is claimed is:
 1. A method for reducing likelihood of infusionreactions during PEGylated uricase therapy in a gout patient withoutmeasuring anti-PEGylated uricase and anti-PEG antibody titers,comprising the steps of: intravenously administering to said patient atherapeutically effective amount of PEGylated uricase every two weeks;obtaining a biological sample from said patient after a period of atleast 2 weeks, wherein said biological sample is selected from the groupconsisting of blood, serum and plasma; measuring a uric acid level insaid biological sample; and indicating that the uric acid level isassociated with a lower likelihood of antibody-mediated PEGylateduricase clearance when said uric acid level is maintained at less than 6mg/dl or indicating that said uric acid level is associated with ahigher likelihood of antibody-mediated PEGylated uricase clearance at atime point when said uric acid level is measured at least 6 mg/dl; ifsaid uric acid level is less than 6 mg/dl then continuing saidadministration of PEGylated uricase every two weeks; or if said uricacid level is above 6 mg/dl then discontinuing said administration ofPEGylated uricase every two weeks; wherein said patient previouslyreceived ineffective allopurinol therapy or has a medical history inwhich allopurinol therapy is contraindicated.
 2. The method according toclaim 1, wherein said gout is refractory.
 3. The method according toclaim 1, wherein said gout is chronic or tophaceous.